Coactivator-independent vitamin D receptor signaling causes severe rickets in mice, that is not prevented by a diet high in calcium, phosphate, and lactose

The vitamin D receptor (VDR) plays a critical role in the regulation of mineral and bone homeostasis. Upon binding of 1α,25-dihydroxyvitamin D3 to the VDR, the activation function 2 (AF2) domain repositions and recruits coactivators for the assembly of the transcriptional machinery required for gene transcription. In contrast to coactivator-induced transcriptional activation, the functional effects of coactivator-independent VDR signaling remain unclear. In humans, mutations in the AF2 domain are associated with hereditary vitamin D-resistant rickets, a genetic disorder characterized by impaired bone mineralization and growth. In the present study, we used mice with a systemic or conditional deletion of the VDR-AF2 domain (VdrΔAF2) to study coactivator-independent VDR signaling. We confirm that ligand-induced transcriptional activation was disabled because the mutant VDRΔAF2 protein was unable to interact with coactivators. Systemic VdrΔAF2 mice developed short, undermineralized bones with dysmorphic growth plates, a bone phenotype that was more pronounced than that of systemic Vdr knockout (Vdr−/−) mice. Interestingly, a rescue diet that is high in calcium, phosphate, and lactose, normalized this phenotype in Vdr−/−, but not in VdrΔAF2 mice. However, osteoblast- and osteoclast-specific VdrΔAF2 mice did not recapitulate this bone phenotype indicating coactivator-independent VDR effects are more important in other organs. In addition, RNA-sequencing analysis of duodenum and kidney revealed a decreased expression of VDR target genes in systemic VdrΔAF2 mice, which was not observed in Vdr−/− mice. These genes could provide new insights in the compensatory (re)absorption of minerals that are crucial for bone homeostasis. In summary, coactivator-independent VDR effects contribute to mineral and bone homeostasis.


INTRODUCTION
Active vitamin D, 1α,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ], is a key regulator of calcium and phosphate (re)absorption and is crucial for calcium, phosphate, and bone homeostasis.The vitamin D precursor, 25(OH)D 3 , is converted into 1,25(OH) 2 D 3 by 1αhydroxylase that is encoded by Cyp27b1.1,25(OH) 2 D 3 exerts its functions through binding to the vitamin D receptor (VDR; NR1I1), which regulates gene expression by acting as a ligand-induced transcription factor. 1 Upon binding of 1,25(OH) 2 D 3 to the ligand binding domain (LBD) of the VDR, the VDR undergoes conformational changes that evoke the association with the retinoid X receptor (RXR) and the repositioning of the carboxyterminal helix 12, which contains the activation function 2 (AF2) domain of the VDR.Within the nucleus, the VDR-RXR heterodimer binds to vitamin D responsive elements (VDRE) in the genome via two conserved zinc fingers in its amino terminal DNA-binding domain (DBD).2][3] Alternatively, the VDR is able to repress gene transcription via interaction of its LBD with corepressors.However, as X-ray crystal structures of corepressor peptides bound to VDR have not been reported, the exact location at which corepressors bind the VDR remains unclear, but is suggested to be independent of the AF2 domain.Interaction of the VDR with corepressors reduces its transcriptional activity by disrupting its interaction with transcription-promoting complexes and by tightening the chromatin structure. 4nterestingly, in mice and humans, inactivating mutations in CYP27B1 and VDR result in hypocalcemia, hypophosphatemia, secondary hyperparathyroidism and short, deformed bones with dysmorphic growth plates.][7][8][9][10] In mice and humans, functional mutations in CYP27B1 do not result in alopecia, while VDR mutations that disrupt its DNA binding do cause alopecia that is not reversed or prevented by a diet high in calcium, phosphate, and lactose.Thus, the presence of a functional VDR in absence of 1,25(OH) 2 D 3 is sufficient to maintain hair follicle homeostasis. 11][18][19] Clearly, the VDR has the ability to repress important genes and pathways.However, continuous coactivator-dependent transcriptional induction by physiological levels of 1,25(OH) 2 D 3 likely hinder the detection of targets that can also be repressed by the VDR.We hypothesize that the interaction of the VDR with corepressors is largely independent of its interaction with coactivators, and that we can reveal novel VDR targets by disrupting its interaction with coactivators.Therefore, in the present study, we used a transgenic mouse model expressing a VDR that lacks its AF2 domain (Vdr ΔAF2 ). 20We performed a detailed study of the bone phenotype of this mouse model and compared it to that of Vdr −/− mice, which have lost both transcriptional induction and repression.

RESULTS
No coactivator-but preserved corepressor interaction with VDR ΔAF2 after 1,25(OH) 2 D 3 binding To map interactions of the wild-type VDR (VDR +/+ ) and truncated VDR ΔAF2 proteins with coregulatory proteins, a chip platform for nuclear receptor activity profiling (NAPing) was used.The genomic mutation in Vdr −/− mice resulted in a short VDR protein that was quickly degraded (Fig. S1).Therefore, this condition was not included in the NAPing assay.As expected, 1,25(OH) 2 D 3 induced a shift in the coregulatory profile for VDR +/+ , resulting in a significantly increased interaction with nuclear receptor coactivators (e.g.NCOAs), whereas interaction with nuclear receptor corepressors (e. g.NCOR1) was significantly downregulated (Fig. 1a, b).In contrast, 1,25(OH) 2 D 3 did not induce coactivator binding to the VDR ΔAF2 protein, further proving the importance of the AF2 domain for coactivator binding.Interestingly, interaction of VDR ΔAF2 with the corepressor NCOR1 tended to be elevated in response to 1,25(OH) 2 D 3 stimulation (Fig. 1a, b).Correspondingly, because of its inability to interact with coactivators, the VDR ΔAF2 protein was unable to transactivate a VDRE-containing reporter construct in an in vitro transfection assay (Fig. 1c).These data demonstrate that the VDR ΔAF2 protein is unable to activate gene transcription but suggest it can still exert repressive effects.
]21 In Vdr ΔAF2 mice on the other hand, the VDR ΔAF2 protein still interacts with corepressors, as shown by our NAPing assay, maintaining the possibility of transcriptionally repressing VDR target genes.To explore the phenotypical consequences of losing transcriptional induction but maintaining transcriptional repression, we compared 8-week-old Vdr ΔAF2 mice to 8-week-old Vdr −/− mice on a normal diet (1% calcium, 0.7% phosphate) and on a rescue diet (2% calcium, 1.25% phosphate, 20% lactose).
On the normal diet, body weight and tibia length of Vdr −/− mice were significantly reduced compared to Vdr +/+ littermates (Fig. 2a, b).However, body weight and tibia length of Vdr ΔAF2 mice were even lower than that of Vdr −/− mice (Fig. 2a, b).In addition, body weight and tibia length of Vdr −/− were almost completely corrected by the rescue diet, whereas these parameters could not be normalized in Vdr ΔAF2 mice (Fig. 2c, d).Of note, unlike Vdr −/− mice which started to develop alopecia from 8 weeks of age, Vdr ΔAF2 mice did not develop alopecia (Fig. S2).Clearly, phenotypic abnormalities were more pronounced in Vdr ΔAF2 mice compared to Vdr −/− mice, suggesting that coactivator-independent VDR signaling/repression is even more deleterious than complete absence of genomic VDR signaling.Calcium, phosphate, and bone homeostasis are more severely impaired in Vdr ΔAF2 than in Vdr −/− mice On a normal diet, Vdr −/− mice were hypocalcemic, hypophosphatemic, had elevated parathyroid hormone (PTH) levels and virtually undetectable fibroblast growth factor (FGF23) levels, while urinary fractional excretion of calcium and phosphate remained relatively high given the hypocalcemia and hypophosphatemia (Fig. 3a, b).Vdr ΔAF2 mice had similar serum phosphate, PTH, and FGF23 levels as observed in Vdr −/− mice, whereas serum calcium was significantly lower compared to Vdr −/− and Vdr +/+ mice (Fig. 3a, b).Interestingly, feeding a rescue diet normalized serum calcium, phosphate, PTH, and largely normalized FGF23 levels in Vdr −/− mice (Fig. 3c), whereas fractional excretion of calcium and phosphate were slightly elevated (Fig. 3d).In contrast, Vdr ΔAF2 mice on this rescue diet remained hypocalcemic and hypophosphatemic and had highly elevated serum PTH and undetectable FGF23 levels, while fractional excretion of calcium and phosphate was similar to Vdr +/+ mice (Fig. 3c, d).
We next evaluated the bone phenotype of Vdr −/− and Vdr ΔAF2 mice by micro-computed tomography (µCT) analysis.When fed a normal diet, tibiae of Vdr −/− mice showed extreme trabecularization of the metaphysial region, significantly increased cortical porosity and cross-sectional tissue area without changes in cortical thickness, compared to Vdr +/+ mice (Fig. 4a, b).The amount of calcium per femur dry weight, a measure of bone mineralization, was significantly decreased in Vdr −/− mice compared to Vdr +/+ mice (Fig. 4c).Interestingly, on the normal diet, Vdr ΔAF2 mice had more pronounced metaphyseal trabecularization and significantly lower cortical thickness compared to Vdr −/− mice (Fig. 4a, b).The amount of calcium per femur dry weight was also significantly lower in Vdr ΔAF2 mice than in Vdr −/− mice (Fig. 4c).Von Kossa and calcein stainings visually confirmed that mineralization of the tibial bones was reduced in Vdr −/− and especially in Vdr ΔAF2 mice compared to Vdr +/+ mice (Fig. 4d).The rescue diet largely prevented the bone phenotype of Vdr −/− mice.Indeed, trabecularization of the metaphysical region was absent in Vdr −/− mice on the rescue diet, and trabecular bone mass (data not shown) and cortical porosity were similar to that of Vdr +/+ mice (Fig. 4e, f).However, on this rescue diet cortical thickness remained significantly lower in Vdr −/− mice than in Vdr +/+ littermates.Interestingly, although calcium and phosphate deposition in bone was markedly improved on the rescue diet (Fig. 4g, h), Vdr ΔAF2 mice persistently showed an aberrant bone phenotype including enlarged, undermineralized metaphysis and increased cortical porosity (Fig. 4e-h).
Histological characterization of tibial bones confirms that bone homeostasis is more severely impaired in Vdr ΔAF2 than in Vdr −/− mice To better understand the differences in bone phenotype between Vdr ΔAF2 and Vdr −/− mice, an extensive histological analysis was performed.
Goldner staining revealed large areas of unmineralized bone matrix (osteoid) in Vdr −/− and Vdr ΔAF2 mice on the normal diet.This excessive osteoid deposition was largely normalized by the rescue diet, although still present in Vdr ΔAF2 mice (Fig. 5a).Masson trichrome staining illustrated that the large unmineralized bone areas in Vdr −/− and Vdr ΔAF2 mice on the normal diet and in Vdr ΔAF2 mice on the rescue diet mainly consisted of collagen (Fig. 5b).In addition, the Masson trichrome and Safranin-O staining clearly showed the growth plate abnormalities previously described in Vdr −/− mice fed a normal diet.These growth plate abnormalities were persistent in Vdr ΔAF2 mice, but not in Vdr −/− mice on the rescue diet (Fig. 5b, Fig. S3). 10,22Hematoxylin and eosin (H&E) staining revealed osteoblasts and fibroblast-shaped cells filling the bone marrow spaces between the undermineralized bone in Vdr −/− and Vdr ΔAF2 mice, which persisted in the latter when given a rescue diet (Fig. 6a).Indeed, immunofluorescent labeling of osterix (Osx), an osteoblast marker, and vimentin, a mesenchymal cell marker highly expressed in fibroblasts, confirmed the presence of osteoblasts and fibroblasts, occupying the spaces between the undermineralized bone (Fig. 6b).In addition, serum Vdr ΔAF2 Vdr ΔAF2 Vdr +/+ Vdr -/- osteocalcin levels, a marker for osteoblast activity, were increased in Vdr ΔAF2 mice on the rescue diet (Fig. 6c).On the other hand, total osteoclast surface over bone surface (OC.S/BS) tended to be decreased in Vdr −/− and Vdr ΔAF2 mice on the normal diet, although, the (undermineralized) bone volume over tissue volume [(BV/TV)/%] was also significantly increased in both strains compared to Vdr +/+ littermates (Fig. 6d, e).On the rescue diet, no differences in OC.S/BS were observed, whereas BV/TV remained significantly increased in Vdr ΔAF2 mice compared to Vdr −/− and Vdr +/+ mice (Fig. 6d, e).However, serum carboxyterminal collagen crosslinks (CTx) levels, a marker for osteoclast activity, were only significantly increased in Vdr ΔAF2 mice on the normal diet but not in Vdr ΔAF2 mice on the rescue diet (Fig. 6f).
Together, these findings suggest that coactivator-independent VDR signaling negatively affects mineral and bone homeostasis to a greater extent than absence of genomic VDR signaling, and that these effects cannot be completely prevented by feeding a diet high in calcium, phosphate, and lactose.

No manifest role for coactivator-independent VDR signaling in bone cells
To differentiate direct actions of coactivator-independent VDR signaling within bone from its systemic effects on mineral supply to bone, we generated mice expressing the mutant VDR only in osteoblasts or osteoclasts by crossing paired related homeobox 1 (Prrx1)-Cre or M lysozyme (LysM)-Cre mice with Vdr lox/ΔAF2 mice, respectively, and compared their phenotype with osteoblast-and osteoclast-specific Vdr −/− mice, generated by crossing Prrx1-Cre or LysM-Cre mice with Vdr lox/lox mice.We first validated the efficacy of the Cre-recombinase driven by the Prrx1 promoter.In primary osteoblast cultures from Prrx1-Cre + ;Vdr lox/lox mice, Vdr gene and VDR protein expression was significantly reduced (almost complete knockdown) compared to osteoblasts derived from Prrx1-Cre-;Vdr lox/lox littermates (Fig. 7a, b).Treatment of these cells with 1,25(OH) 2 D 3 induced the expression of Cyp24a1, a primary VDR target gene, although the level of induction was much more pronounced in Prrx1-Cre -;Vdr lox/lox mice than in Prrx1-Cre + ;Vdr lox/lox mice (Fig. 7c).Prrx1-Cre mediated deletion of Vdr expression was specific to osteochondrogenic lineage cells, as Vdr expression was, next to osteoblasts, significantly decreased in chondrocytes isolated from Prrx1-Cre + ;Vdr lox/lox mice compared to those isolated from Prrx1-Cre -;Vdr lox/lox mice.Vdr expression was unaltered in other Vdr-target tissues such as, kidney, duodenum, colon, white adipose tissue, and brown adipose tissue (Fig. 7d).To confirm that the introduction of only one Vdr ΔAF2 allele was sufficient to exert coactivator-independent effects, systemic Vdr -/ΔAF2 mice were compared to systemic Vdr +/ΔAF2 and Vdr ΔAF2/ΔAF2 mice.Both Vdr -/ΔAF2 and Vdr ΔAF2/ΔAF2 mice displayed the same rickets-like bone phenotype, whereas mice containing at least one Vdr + allele had a normal bone phenotype (Fig. 7e).Body weights as well as tibia lengths were unaltered in both Prrx1-Cre + ;Vdr lox/lox and Prrx1-Cre + ;Vdr lox/ΔAF2 mice compared to their Cre-littermates (Fig. 7f).Serum calcium, phosphate, PTH, and FGF23 levels were unaltered in both Prrx1-Cre + ;Vdr lox/lox and Prrx1-Cre + ;Vdr lox/ΔAF2 mice compared to their Cre-littermates (Fig. 8a).Fractional excretion of calcium and phosphate was also normal (Fig. 8b).However, trabecular and cortical bone mass, assessed by µCT analysis, was similarly increased in Prrx1-Cre + ;Vdr lox/lox and Prrx1-Cre + ;Vdr lox/ΔAF2 mice compared to their Cre -littermates, as evidenced by increased BV/TV, increased trabecular number and thickness, and increased cortical thickness (Fig. 8c, e).Although trabecular and cortical bone mass was increased, bone calcium content measured in ashed femurs remained unaltered, as well as serum osteocalcin and CTx levels (Fig. 8f).Osteoblast numbers, assessed on H&E staining, OC.S/ BS, assessed on tartrate resistant acid phosphatase (TRAP) staining, and the mineral opposition rate (MAR), assessed by analysis of calcein labels were similar in both strains (Fig. 8g).Finally, gene expression levels measured in femurs of Prrx1-Cre;Vdr lox/lox and Prrx1-Cre;Vdr lox/ΔAF2 mice also remained largely unaltered (Fig. S4).
These findings refute the idea that coactivator-independent signaling of the VDR has important direct effects in skeletal cells themselves and therefore suggest a more important role in intestine and kidney.

Transcriptomic analysis suggests promising target genes of VDRmediated repression involved in mineral ion transport
To investigate differences in transcriptional regulation that could (at least in part) account for the more severe phenotype of Vdr ΔAF2 mice compared to Vdr −/− mice, RNA-sequencing (RNA-seq) studies on duodenum and kidney of Vdr +/+ , Vdr −/− and Vdr ΔAF2 mice were performed.In duodenum, the expression of 1 389 genes was significantly (P < 0.05) changed (702 up; 687 down) when comparing Vdr ΔAF2 to Vdr +/+ mice.When comparing Vdr ΔAF2 to Vdr −/− mice, 1 329 genes were differentially regulated (743 up; 586 down) (Fig. 10a).Similar numbers of differentially expressed genes were observed in kidney when comparing Vdr ΔAF2 to Vdr +/+ mice (881 up; 939 down) or to Vdr −/− mice (693 up; 647 down) (Fig. 10b).To evaluate the difference between the complete loss of genomic VDR signaling and the loss of coactivator-dependent signaling, we further focused our analysis on the comparison between Vdr −/− and Vdr ΔAF2 mice.Of the resulting list of significantly differentially regulated genes (Fig. 10c, d left panels), we selected the 25 most downregulated genes (Fig. 10c, d right panels).On top of this gene list, we found Trpv6 (in duodenum) and S100g (in kidney), which are primary VDR target genes and important regulators of calcium homeostasis (Fig. 10c, d).Other genes in this list include genes involved in mineral ion transport (Trpv6, Car7, Cabp1 and Wdr72 in duodenum and S100g, Slc22a27 and Slc13a1 in kidney) and in fatty acid transport (Bbox1 in kidney).To validate these RNA-seq results (Fig. 10a, b), well known VDR target genes [duodenum (Trpv6) and kidney (Cyp24a1, S100g, Cyp27b1)] were selected and verified by quantitative polymerase chain reaction (qPCR) (Fig. 10e, f).

DISCUSSION
VDR expression in intestine and kidney is crucial to maintain an adequate mineral supply to serum and to bone.However, some important VDR target genes and pathways involved in mineral transport may remain undetected due to the continuous coactivator-dependent transcriptional induction.Therefore, we used a transgenic mouse model expressing a VDR that lacks its AF2 domain (Vdr ΔAF2 ). 20The NAPing assay, performed on the fulllength VDR +/+ and VDR ΔAF2 protein, confirmed that the VDR ΔAF2 was completely unresponsive to ligand-induced interaction with coactivators.This demonstrates that coactivator binding to the VDR is completely dependent on a functional AF2 domain.In contrast, co-repressor interaction with the VDR is AF2 domainindependent. 23This observation suggests that the VDR ΔAF2 may function as a transcriptional repressor, a function that has been described for unliganded nuclear receptors, such as TR and PPAR. 16,19However, ligand-independent repression is most likely not identical to AF2-independent repression, as some VDR target genes are repressed in a ligand-dependent manner. 24The Vdr ΔAF2 independent but corepressor-dependent, repressive VDR signaling.6][27] Here, we show that the effects of coactivatorindependent VDR signaling extend beyond those observed in hair follicle homeostasis, 14,28 evidenced by the more severely impaired mineral homeostasis and bone phenotype observed in Vdr ΔAF2 mice compared to Vdr −/− mice.We hypothesized that VDR ΔAF2mediated repression has direct negative effects on skeletal cell function and/or inhibits mineral absorption.We next targeted Vdr expression in osteoblast lineage cells by means of Prrx1-promoter driven recombination, which reduces both osteoblastic and chondrocytic Vdr expression.However, earlier studies demonstrated that chondrocytic Vdr expression has no impact on bone or mineral homeostasis in 8-week-old mice. 29ence, the observed phenotype in Prrx1-Cre + ;Vdr lox/lox mice is attributed to alterations in osteoblastic Vdr expression.Using these osteoblast-specific Vdr −/− and Vdr ΔAF2 mice, we show that the osteoblast-specific loss of VDR signaling or coactivatorindependent VDR signaling results in increased bone mass.In agreement with previous publications, 9,30,31 these data confirm the importance of coactivator-dependent VDR signaling in osteoblasts and shows that coactivator-independent VDR signaling is not important in these cells.Hence, the coactivatorindependent effects responsible for the severe bone phenotype of Vdr ΔAF2 mice are likely its negative effects on mineral (re) absorption, which result in persistent hypocalcemia and hypophosphatemia even on the rescue diet, along with chronically elevated PTH levels.Of note, previous research showed that Vdr −/− and Vdr ΔAF2 mice on the rescue diet have significantly lower circulating levels of 25(OH)D 3 and higher levels of 1,25(OH) 2 D 3 . 20However, both strains are unresponsive to coactivator-dependent 1,25(OH) 2 D 3 signaling.Interestingly, the rescue diet is able to rescue the bone phenotype of Vdr −/− mice but not that of Vdr ΔAF2 mice.Remarkably, renal calcium and phosphate conservation was lower in Vdr −/− mice compared to Vdr ΔAF2 mice on the rescue diet.A possible explanation for this phenotypic difference in renal handling is VDR-mediated repression on calcium and phosphate reabsorption, a function that is lost in Vdr −/− mice.The persistent calcium and phosphate deficits in Vdr ΔAF2 mice likely impair calcium and phosphate deposition into the osteoid matrix produced by osteoblasts.The number of osteoblasts seems to have increased in Vdr ΔAF2 mice as observed on Osx-labeled bone sections.As we did not observe differences in osteoblast numbers in osteoblast-specific Vdr ΔAF2 mice, we assume that external regulators such as PTH are responsible for the observed increased osteoblast numbers in systemic Vdr ΔAF2 mice.Increased osteoblast numbers in turn result in excessive deposition of osteoid matrix and collagen fibers, which remain undermineralized as shown by Goldner, Masson trichrome and Von Kossa/calcein stainings and µCT analysis.In addition, chronically high levels of PTH have been associated with fibrous dysplasia in humans, a disorder where normal bone and bone marrow are replaced by fibrous tissue. 32,33Interestingly, immunohistochemical analysis revealed that this human phenotype resembles the fibroblast-loaded bone phenotype observed in Vdr −/− mice on normal diet and Vdr ΔAF2 mice on both normal and rescue diets.In addition, high PTH levels can induce osteoclastogenesis, as previously reported in Vdr −/− mice on a normal diet.However we did not observe significant changes in OC.S/BS. 34ince these bone defects are likely indirect consequences of the low mineral supply and high PTH levels, we hypothesized that the main effects of coactivator-independent VDR signaling reside in the intestines or kidneys, negatively affecting mineral (re)absorption.Therefore, we performed RNA-seq studies in intestine and kidney of mice on the rescue diet to expose genes that are responsible for the persistent hypocalcemia and hypophosphatemia in Vdr ΔAF2 mice.In our RNA-seq studies, duodenal tissue was used because of the active transcellular calcium absorption and high Vdr expression within this proximal part of the intestine. 8nterestingly, expression of the calcium transporters Trpv6 and S100g was significantly lower in Vdr ΔAF2 than in Vdr −/− mice, suggesting that the VDR does not only induce these transporters but can also repress them.Repression of Trpv6 and S100g might contribute to the more severe hypocalcemia and rickets phenotype observed in Vdr ΔAF2 mice.However, previous studies using Trpv6/S100g double knockout mice have shown that both transporters are important but not crucial factors in maintaining normal serum calcium levels, suggesting other transporters are to be identified. 35,36Therefore, we searched for other genes that might contribute to the observed phenotype in Vdr ΔAF2 mice focusing on genes that resembled the expression pattern of Trpv6 and S100g, which were significantly more downregulated in Vdr ΔAF2 than in Vdr −/− mice.We identified genes involved in mineral ion transport (Car7, Cabp1, and Wdr72 in duodenum; Slc22a27 and Slc13a1 in kidney) and will investigate the physiological relevance of these genes in the future.
In conclusion, deletion of the VDR AF2 domain completely impairs ligand-dependent coactivator binding, but retains AF2independent co-repressor binding.Here, we demonstrate the negative impact of these repressive actions in duodenum and kidney and its implications on calcium, phosphate, and bone homeostasis, evidenced by the more extremely impaired mineral homeostasis and bone phenotype observed in Vdr ΔAF2 mice compared to that in Vdr −/− mice.We demonstrate that this bone phenotype is not mediated by coactivator-independent actions directly in bone cells, further emphasizing its role in intestine and kidney.
In addition, the Vdr ΔAF2 phenotype is also largely unresponsive to a diet high in calcium, phosphate, and lactose.Finally, based on this mouse model, we propose a list of potentially important, new, repressed VDR target genes in the interrelation between inorganic ions and calcium/phosphate (re)absorption.Trans-repression assays are currently being developed in our lab to further assess repressive VDR signaling in vitro.

Transgenic mouse models
Vdr ΔAF2 mice were a kind gift of Dr. S. Kato (Institute of Molecular and Cellular Biosciences, University of Tokyo, Soma Central Hospital, Japan), 20 and these mutant mice were obtained by introducing two stop codons at the beginning of exon 10, thereby transcriptionally deleting the 12 most C-terminal amino acids, comprising the AF2 domain of the VDR.Vdr −/− mice were purchased from the Jackson laboratory (JAX stock #006133). 10hese mice were generated by replacing exon 3, encoding the second zinc finger of the VDR, with a neomycin resistance gene.Genomic DNA of the three different mouse strains was sent for whole genome sequencing (Novogene, United Kingdom) to confirm the pre-determined mutations and to exclude off-target mutations.
Phenotypic analysis was performed on 8-week-old Vdr −/− , Vdr ΔAF2 , and their Vdr +/+ littermates.Only female mice were included in this study as preliminary data on calcium and bone homeostasis showed no differences between male and female mice (data not shown).The genetic background of the mouse strains was characterized by a 384-SNP panel [Mouse Max Bax 384 SNP Panel (GM-SN-15), Charles River Genetic Testing Services Wilmington] on genomic DNA from tail cuts.Both strains were C57BL/6 J congenic ( ≥ 99.9% C57BL/6 J allelic profile percent match).Therefore, Vdr +/+ mice of both strains were pooled into one group.Genotyping was performed by polymerase chain reaction (PCR) with Go Taq G2 Flexi DNA polymerase (Promega) on genomic DNA from toe cuts.Heterozygous breeding pairs were phenotypically normal (data not shown) and maintained on a mouse breeding diet (V1124, Ssniff, Soest, Germany), whereas experimental mice were either weaned on a normal diet [1% calcium, 0.7% phosphate, 0% lactose (V1535, Ssniff)] or a high calcium diet (2% calcium, 1.25% phosphate, 20% lactose, Teklad custom diet TD.96348, Inotiv, West Lafayette, US), the latter referred to as "rescue diet".
To analyze calcium apposition in bone, calcein (16 mg/kg body weight; Merck) was administered via intraperitoneal injections 4 days and 1 day prior to sacrifice.All mice were housed in an animal facility with 12 h dark/light cycles and constant room temperature with food and water supplied ad libitum.All animal experiments were approved by the ethical committee of the KU Leuven (P188/2016).
Serum and urine biochemistry At 8 weeks of age mice were transferred to individual metabolic cages (Tecniplast, Buguggiate, Italy) to obtain 24 h urine collections and monitor food and water intake.Serum was collected at sacrifice.Urine and serum calcium (OSR60117), phosphate (OSR6122), and creatinine (OSR6178) concentrations were measured with a Beckman Colter DxC700AU chemistry analyzer (Analis, Suarlée, Belgium) and fractional urinary excretion of calcium and phosphate was calculated based on serum and urine calcium, phosphate, and creatinine levels.Serum concentrations of PTH (Quidel, San Diego, USA), FGF23 (Kainos,Tokyo, Japan), and CTx (Ratlaps, IDS, Frankfurt, Germany) were measured with an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions.Serum osteocalcin levels were measured using an in-house radio-immunoassay. 39cro-computed tomography (µCT) The high resolution SkyScan 1272 system (Bruker, Belgium) was used to obtain ex vivo µCT images of the right tibia (source settings; 60 kV, 83 μA, 0.5 mm aluminum filterscan settings; 5 µm pixel size, 180°a ngular rotation, 0.4°angular increment).Cone-beam reconstruction software (NRecon, Bruker) was used to reconstruct the scans based on the Feldkamp algorithm.These reconstructed datasets were used for 3D morphometric analysis using CT Analyzer software (CTAn, Bruker).Volumes of interest were selected at 2.25 mm -2.75 mm (cortical) and 0.75 mm -2 mm (trabecular) from a manually selected reference point beneath the growth plate, where the trabecular compartments converge into one compartment on a cross-sectional image.In systemic Vdr −/− and Vdr ΔAF2 mice, distances from the reference point were adapted to the average tibia length of the strain to compensate for the large differences in tibia length between the strains (Table S1).The "automated trabecular and cortical bone selection method" (Bruker Method Note 008) was adapted and used to automatically select cortical regions of interest within the volumes of interest.Binary images were generated using a global thresholding of 80-255 determined to optimally separate bone and soft tissue.Analysis of these binary images was performed according to the guidelines of the American Society for Bone and Mineral Research 40 and 3D models were constructed with CTvox software (Bruker, Belgium).Extreme trabecularization of the metaphyseal region disabled correct selection and analysis of the trabecular bone compartment in Vdr −/− and Vdr ΔAF2 mice on the normal diet and in Vdr ΔAF2 mice on the rescue diet.
Femur calcium content Femurs were dried overnight at 100 °C and subsequently ashed for 5 h at 500 °C.Femur dry and ash weights were quantified.Ashes were dissolved overnight in 1 mL 1 mol/L HCl and diluted 1:50 in distilled water to measure the calcium concentration on a Beckman Colter DxC700AU chemistry analyzer.

Fig. 2
Fig. 2 Phenotypic comparison of ligand-independent Vdr ΔAF2 and receptor-independent Vdr −/− mice.Normal diet (upper panel) and rescue diet (bottom panel).a, c Representative pictures of mice and quantification of body weights and b, d representative pictures of tibiae and quantification of their lengths measured in 8-week-old female mice (n ≥ 8)

Fig. 3 (
Fig. 3 Calcium and phosphate homeostasis in Vdr +/+ , Vdr −/− and Vdr ΔAF2 mice.a, c Serum calcium, phosphate, PTH and FGF23 levels and b, d fractional excretion of calcium and phosphate /% of 8-week-old female mice weaned on a a, b normal diet or on a c, d rescue diet Fig. 4 The rachitic bone phenotype of Vdr ΔAF2 mice is not prevented by a rescue diet high in calcium and phosphate.a, e Representative µCT images of the sagittal plane and cortical section of tibial bones, where red dots depict the reference point and blue boxes depict the analyzed cortical volume of interest.b, f Quantification of cortical bone parameters (cross-sectional tissue area, porosity, and cortical thickness).c, g Absolute calcium per femur dry weight.d, h Representative images of Von Kossa stained and calcein labeled -tibiae.All parameters are measured in 8-week-old, female Vdr +/+ , Vdr −/− and Vdr ΔAF2 mice on normal (upper panel) or rescue diet (lower panel)

aFig. 6
Fig. 6 Histochemical and immunofluorescent comparison of Vdr +/+ , Vdr −/− and Vdr ΔAF2 mice; H&E, Osx-vimentin co-staining and TRAP staining.Representative images of metaphyseal regions on a H&E-stained sections (osteoblasts, and fibroblasts shaped cells are depicted by blue and green arrowheads, respectively) and b immunofluorescence using anti-Osx (blue) and anti-vimentin (green) antibodies.Cell nuclei are stained with Hoechst (blue).c Serum osteocalcin levels.d Representative overview images of TRAP stained sections and e quantification of (Oc.S/BS)/% and bone area/μm 2 and f Serum CTX levels.All stainings and serum analyses were performed on samples from 8-week-old female Vdr +/+ , Vdr −/− and Vdr ΔAF2 mice on normal (upper panels) or rescue diet (lower panels).Scale bars represent 50 µm (a, b) or 500 µm (d)